1. X-linked retinoschisis (XLRS) gene therapy pre-clinical study and clinical trials XLRS is a genetic disease caused by mutations in the retinoschisin (RS1) gene. An absent or mutated RS1 protein in the retina of patients with XLRS leads to abnormalities in the normal laminar structure of the retina, resulting in impaired visual acuity and increased propensity to retinal detachment. Gene replacement therapy holds the promise of curing the disease. In collaboration with Dr. Paul Sieving's group, we have developed and optimized adeno-associated viral (AAV) vectors that are capable of mediating stable and retinal specific expression of human RS1 protein in mice. To meet the FDA requirements for gene therapy clinical trials, we have completed a pre-clinical efficacy study in a retinoschisin knockout mouse model, and a vector toxicology study in both mouse and rabbit models (1-3). Investigational New Drug (IND) application to FDA has been approved and a phase I/II clinical trials are ongoing. As of August 2017, a total of ten XLRS patients have received the vector administration. Data analysis of the trials is being conducted and the results will be published soon. 2. Preclinical gene therapy studies for retinitis pigmentosa due to RPGR or RP2 mutations X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. Gene therapy for RPGR-XLRP was hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the RPGR-ORF15 cDNA that includes a purine-rich 3-coding region. We managed to overcome these problems and have generated AAV vectors carrying full-length mouse and human RPGR ORF15 coding sequences. We have also developed a self-complementary AAV vector carrying human RP2 expression cassette. We have completed proof-of-principle long-term (18-24 months) dose efficacy/toxicity studies in mouse models with RPGR or RP2 deficiency. Our results demonstrate that administration of the RPGR AAV vectors at appropriate doses can significantly preserve the retinal function and delay the photoreceptor loss in the mice with RPGR deficiency. Additionally, administration of the RP2 vector with a broad dose range can remarkable maintain the function and viability of cone photoreceptors in the mice with RP2 deficiency. These results lead to two papers published in Human Molecular Genetics during the 2016 Fiscal Year. In order to bring the therapies to the clinic, during the 2017 Fiscal Year, we have been testing the dose-efficacy-toxicity of an optimized AAV-RPGR vector in two additional mouse models for RPGR disease. The study is about to complete and data analysis is ongoing. We are hoping that IND application of either one of the two therapies to FDA can be filed in one to two years. 3. Gene therapy for Leber congenital amaurosis due to CEP290 mutations Leber congenital amaurosis (LCA) is one of the most common causes of blindness in children. People with this disease typically have severe visual impairment beginning in infancy. Mutations in the CEP290 gene account for 20-25 percent of LCA, afflicting an estimated 20,000 people worldwide. Since the size of CEP290 coding sequence (7.4 kb) exceeds the packaging limit of AAV vector (5kb), we are seeking alternative approaches for treating CEP290-LCA. In particular, we are testing whether delivery of truncated functional domains of Cep290 could complement certain types of CEP290 mutations. We have made a series of AAV vectors carrying different portions of Cep290 coding sequence and have tested them in a mouse model with Cep290 mutation. We recently identified one vector that is capable of preserving the retinal function and structure in the mouse model. A manuscript of this finding will be submitted for publication soon. In addition, we are also testing new approaches to delivery the full-length Cep290 gene and the preliminary results look promising. 4. In vivo application of CRISPR/Cas9 for treating retinal degenerative diseases CRISPR/Cas9 mediated genome editing has been rapidly advancing in recent years. However, its applications in postmitotic photoreceptors-the direct target of a majority of acquired or inherited retinal degeneration, has been limited. We have recently established an AAV-delivered photoreceptor-specific CRISPR/Cas9 system. The system is highly efficient in modifying photoreceptor genome, as exemplified by disrupting EGFP or Nrl gene following vector administration to mice at postnatal day 14 (P14). Ablation of EGFP was achieved in roughly 60 percent of photoreceptors in a mouse model that constitutively expressing EGFP in rods after a single administration of the AAV-CRISPR against EGFP. Insertions and deletions (Indels) occurred in over 90 percent of photoreceptors receiving the AAV-CRISPR against Nrl. As NRL is a transcription factor dictating the rod photoreceptor cell fate, Nrl knockdown in rods resulted in gain of certain cone features and partial loss of rod function, as revealed by results at functional, morphological and molecular levels. We hypothesized that these phenotype changes can make rods survive rod gene mutations and consequently prevent secondary cone degeneration. Our results indeed showed that Nrl knockdown by AAV-delivered CRISPR/Cas9 can rescue retinal degeneration in mice with either recessive or dominant rod gene mutations, suggesting that this strategy could be developed into a viable treatment for retinal degeneration in human. These results have been published in Nature Communications during the 2017 Fiscal Year (4). In order to develop treatment for autosomal dominant retinal diseases, we are also using AAV-CRISPR/Cas9 to directly knockdown genes with dominant mutations in the retina. 5. Core function: AAV vector design and production for collaborating groups During the 2017 Fiscal Year, we have designed and produced about 30 AAV vectors to support the research projects of our own and over ten collaborating groups (5-13).